1. Field of the Invention
The present invention relates to a general process for regenerating soybeans (Glycine max (L.) Merrill) and to plants produced by the process. More particularly, the invention relates to the use of tissue and cell culture for the regeneration of soybeans plantlets for many soybean varieties. The invention also relates to the media used in this process.
2. Description of the Prior Art
Plant regeneration from cells in culture is essential for the application of somatic hybridization, for the production of new varieties through somoclonal variation and for the use of genetic engineering in producing new varieties. Although plants can be regenerated from single cells of a large number of crop species, the efforts with soybean have generally been unsuccessful.
In recent years, plant cell culture successes have has a considerable influence on the respective roles of cell and organism in control of plant growth and development. This concept was supported when isolated plant cells were shown to be amenable to in vitro cultivation and complete plants could be regenerated from cultures derived from somatic cells, either directly via somatic embryogenesis or indirectly via organogenesis. Generally the regeneration pathway of choice is determined empirically by the manipulation of extrinsic factors, espceially growth regulators. Eary investigation of certain plant species have suggested that exogenous auxin concentration is the major factor controlling somatic embryogenesis, such that its reduction leads to the initiation of embryoid formation. In other species, exposure to a definite balance of auxin and cytokinin leads to the formation of organogenesis (shoots, then roots). Such manipulations, however, do not lead to plantlet formation in seed legumes (soybeans).
Despite recent successes in achieving shoot or plantlet formation from explants, callus or suspension cultures of various legumes including alfalfa, clover and Glycine canescens, a wild perennial special related to the soybean, plant regeneration from tissue cultures of soybean (Glycine max) has not been achieved in a general reproducible method which is applicable to many soybean varieties. Shoot or plantlet formation in Glycine max is limited to shoot production from hypocotyl slices, multiplication of cotyledonary buds and an incomplete or aberrant somatic embryogenesis. The embryogenesis from suspension culture proceeds as far as late torpedo stage. Histological examination showed, however, that the embryoids were aberrant and lacked a well-organized shoot apical meristem. Such structures are sometimes termed "neomorphs". One report has been made that plantlet formation from tissue culture was achieved for Glycine max. However, this report appears to have been a random event and not a general method. This report will be discussed further below.
Phillips et al., in Plant Cell Tissue Organ Culture 1, 123 (1981), describe the somatic embryogenesis of soybean in cell suspension or an agar. They utilized hypocotyl or epicotyl tissue for callus initiation on L2 medium. Cell suspension cultures were initiated from callus tissue in SL2 medium. The cell suspension culture could be used to produce globular and heart-shaped embryos or additional callus which could form shoot buds. The formation of somatic embryos or shoot buds was reproducible using basal SL2 or L2 media supplemented with 100 ppm casein hydroylsate, 2.25 .mu.M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 .mu.M abscisic acid (ABA), 0.1 .mu.M 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxymethyl chloride (AMO 1618) and either 15 .mu.M adenine or 0.46 .mu.M kinetin. Although somatic embryos or shoot buds were formed, no plants were obtained for any varities of Glycine max.
Wildholm et al., in Plant Cell Reports 2, 19 (1983), describe the formation of shoots from Glycine canescens callus obtained from hypocotyls or cotyledons. Root formation did not occur, so no plantlets were obtained. The method did not produce shoots when Glycine max (soybean) was the source of the tissue. The formation of shoots from Glycine canescens tissue culture was achieved through callus induction on B5 basal medium containing 0.5 mg/l .alpha.-naphthalene acetic acid (NAA) followed in series by MS basal medium containing 0.5 mg/l of indoleacetic acid (IAA) and 5 mg/l benzyladenine (BA), and finally MS basal medium containing 0.5 mg/l BA.
Gamborg et al., in Plant Cell Reports 2, 209 (1983a), disclose somatic embryogenesis from cell suspension culture in several Glycine species including three cultivars (out of seven tested) of Glycine max. The embryoid induction medium utilized consisted of the major salts of SL, the micronutrients and vitamins of B5, 10 mg/l casamino acids, 15 .mu.M adenine sulfate, 0.2 .mu.M picloram and 0.025-0.25 .mu.M AMO 1618. It was discovered that picloram was necessary for embryo induction and that it could be replaced by 0.5 to 2.0 .mu.M 2,4-D. No embryoids were induced when the auxins NAA, IAA or indole-3-butyric acid (IBA) were utilized in place of the auxins picloram or 2,4-D. After embryoids were induced, they were transferred to embryo growth medium which consisted of SL medium containing various combinations of cytokinins (zeatin or BA), auxins (picloram) and gibberellic acid (GA.sub.3). Embryoids which were formed went to a heart-shaped structure, but failed to develop beyond this stage on the induction medium. Transfer to the growth medium did result in the formation of roots, but shoots were not formed. The use of MS medium or addition of abscisic acid, coconut milk or change in osmolarity did not result in further development.
Gamborg et al., in Plant Cell Reports 2, 213 (1983b), describe the preparation of protoplasts from cell cultures of Glycine tabacina and Glycine soja and from leaf tissue of the soybean Glycine max cultivar (cv.) Williams 82. The protoplasts formed cells which could be induced to form heart-shaped embryos by the procedure of Gamborg et al. (1983a). As in the latter reference, no plantlets were formed from the procedure.
Christianson et al., in Science 222, 632 (1983), disclose the regeneration of a plantlet from cell suspension culture of the soybean Glycine max (L.) Merrill cv. Mitchell. This appears to have been the result of a random event and appears to have resulted from a piece of clonal tissue. Immature embryos were aseptically removed from 2.5-3.0 cm pods and embryo axes were cut into 1-2 mm pieces. These pieces were placed on a solid medium to induce callus formation. This medium consisted of MS salts, 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine, 100 mg/l thiamine, 100 mg/l inositol, 2% sucrose and 5 mg/l 2,4-D. The hard, non-friable tissue was selected for transfer to new medium and resulted in a tissue line that gave rise to hard, green, glossy, abnormal embryos. When the callus tissue was transferred from the induction medium to an N-amended medium and then transferred back to the induction medium, one exceptional piece of tissue was obtained which was covered with small embryoids. The N-amended medium consisted of the induction medium in which the two nitrogen salts of the MS salts were replaced with 20 mM ammonium citrate. Transfer of the embryoids to a medium containing 0.005 mg/l IBA and 0.2 mg/l BA gave rise to shoot formation. Transfer of the shoots to a basal medium containing 0.1 mg/l of IAA resulted in root formation to produce plantlets. This procedure does not appear to be generally applicable for regenerating soybean cultivars. Instead, it appears to have been a random event which may not be reproducible. Support for this analysis lies in the source of the embryogenic tissue. Christianson et al. state that "one exceptional piece of tissue" with embryoids was obtained. Since only one was obtained out of many initiated, it implies that this was a random event and could have been clonal in nature.
The prior art does not describe a procedure for the regeneration of the soybean Glycine max from tissue and cell culture which is general and reproducible. The prior art does not describe a procedure which results in the formation of regenerative embryoids from the majority of calli induced instead of from a single event with a single callus. The present invention is the first instance of a broadly and generally applicable procedure for regenerating cultivars of the soybean Glycine max.
Soybean plants and seeds are produced by this process. The soybean plants resulting from this process may differ from the starting plant material as a result of somoclonal variation. The pathway is also useful in that it will enable the use of various selection processes to provide further variation. The plants which are produced can be used in conventional breeding programs.